Experimental proof for the structure of a thrombin-inhibiting heparin molecule.

Kinetic studies of thrombin inhibition by antithrombin in the presence of heparin have shown that thrombin binds to heparin in a preformed heparin-antithrombin complex. To study the relative position of the thrombin binding domain and the antithrombin binding domain on a heparin molecule we have designed and synthesized heparin mimetics, which structurally are very similar to the genuine polysaccharide. Their inhibitory properties with respect to factor Xa and thrombin provide experimental evidence that in heparin the thrombin binding domain must be located at the nonreducing end of the antithrombin binding domain to observe thrombin inhibition. As expected, factor Xa inhibition is not affected by elongation of the antithrombin binding pentasaccharide sequence, regardless of the position in which this elongation takes place.

Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.

A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.

Identification of a hexasaccharide sequence able to inhibit thrombin and suitable for 'polymerisation'.

Three hexasaccharides, having from low to very high affinity for antithrombin, were synthesised from disaccharide building block precursors. One of them, methyl(sodium 2,3-di-O-methyl-4-O- sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]2-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside, obtainable from a single disaccharide building block precursor, constitutes a good starting point for obtaining simple oligosaccharidic heparin mimetics able to inhibit the two coagulation factors thrombin and Factor Xa.

Assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition.

Deca- to eicosasaccharides having the generic structure methyl(sodium 2,3-di-O-methyl-4-O-sodium sulfonato-alpha-L-idopyranosyluronate)-(1-->4)-[(2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranosyl)-(1-->4)-(sodium 2,3-di-O-methyl-alpha-L-idopyranosyluronate)-(1-->4)]n-2,3,6-tri-O-sodiu m sulfonato-alpha-D-glucopyranoside have been synthesized from a single disaccharide precursor. All of them bind to and activate antithrombin. When n < or = 6 only Factor Xa inhibition is observed, whereas when n > 6 Factor Xa and thrombin are both inhibited in the presence of antithrombin. These results indicate that, in heparin, the sequence involved in antithrombin-catalyzed thrombin inhibition is a pentadeca- or a hexadecasaccharide.

Synthesis of a 3-deoxy-L-iduronic acid containing heparin pentasaccharide to probe the conformation of the antithrombin III binding sequence.

We report in this work the total synthesis of a close analogue of the pentasaccharide active site of heparin, in which the L-iduronic acid residue has been deoxygenated at position three. 1H NMR studies demonstrated that, as anticipated, such a modification induces a shift of the conformational equilibrium toward 1C4 (contribution to the conformational equilibrium rises from 37% to 65%) and a substantial decrease of the affinity for antithrombin III (Kd 0.154 microM versus 0.050 microM).

Combined NMR and molecular modeling study of an iduronic acid-containing trisaccharide related to antithrombotic heparin fragments.

An iduronic acid-containing trisaccharide, methyl-O-(4-O-methyl-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1-->4)-O- (2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-O-2,6-di-O-sulfo-alpha-D-glucopyranoside, related to antithrombotic heparin fragments has been subjected to a combined NMR and molecular modeling investigation. The conformational behavior of the two constituting disaccharide segments was investigated using a systematic grid search approach with the MM3 force field along with the proper parameters for the sulfate ester group. The exploration of the potential energy surfaces of the trisaccharide was performed through the use of the CICADA methods interfaced with the MM3 force field. In all cases, the 2-O-sulfo-alpha-L-iduronate moiety was given the three favored ring conformations (1)C4, (4)C1, and (2)S0. Conformations were clustered into families, four of which are likely to exhibit significant occupancy in solution. The different low-energy conformational families display different orientations at the glycosidic linkages and/or different ring shapes for the iduronate ring. The (2)S0 conformation is the major one for the 2-O-sulfo-alpha-L-iduronate but is still in equilibrium with the (1)C4 ring shape. The occurrence of such a conformational equilibrium in solution was probed via high-resolution NMR spectroscopy through measurements of coupling constants and NOE build-up. These results are in keeping with the observation that 2-O-sulfated pentasaccharides display a similar affinity for antithrombin III as their 2-N-sulfated counterparts.

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel.

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an alpha-helix stretch (residues 5-12). This alpha-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an alpha-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional 1H nuclear magnetic resonance and show that 1) the proline residue does not disturb the alpha-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an alpha-helix running from residues 5 to 12, two antiparallel stretches of beta-sheet (residues 15-20 and 23-27) connected by a type I' beta-turn, and three disulfide bridges connecting the alpha-helix to the beta-sheet.

Determination of the structure of [Nle7]-endothelin by 1H NMR.

[Nle7]-endothelin was synthesized and studied by 1H NMR and distance geometry calculations. The NMR study was performed first in DMSO-d6 and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two-dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three-dimensional structure of [Nle7]-endothelin is proposed and discussed.

(1)H NMR Study of the solution structure of sarafotoxin-S6b.

Sarafotoxin-S6b has been synthesized and studied by (1)H NMR in 50 50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. On the basis of these data, a 3D structure of sarafotoxin is proposed and compared to that of [Nle(7)]endothelin obtained in the same conditions. From this study, it appeared that sarafotoxin-S6b and [Nle(7)]endothelin roughly share the same 3D structure, the main differences being located in the 4-7 loop bearing the sequence variation.